Methods and compositions for cloning nucleic acid molecules

ABSTRACT

The present invention is directed generally to methods facilitating the cloning of nucleic acid molecules. In particular, the invention relates to the use of polymerase inhibitors, including but not limited to anti-polymerase antibodies (such as anti-Taq antibodies) and fragments thereof, to inactivate residual polymerase activity remaining after the amplification (particularly via PCR) of a target nucleic acid molecule. The invention further provides compositions, particularly storage-stable compositions, comprising one or more components, such as one or more restriction endonucleases and one or more polymerase inhibitors, that are useful in cloning amplified or synthesized nucleic acid molecules by the above-described methods. The invention also relates to nucleic acid molecules produced by these methods, and to genetic constructs (such as vectors) and host cells comprising these nucleic acid molecules.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/055,849, filed Aug. 15, 1997, the disclosure of which is entirelyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the fields of molecular and cellular biology. Theinvention is generally directed to amplification of nucleic acidmolecules and to methods for cloning nucleic acid molecules (DNA or RNA)that have been amplified or synthesized, particularly those nucleic acidmolecules that have undergone PCR amplification. In particular, theinvention concerns methods of cloning amplified nucleic acid moleculescomprising the use of inhibitors of nucleic acid polymerases that carryout the amplification. The invention further concerns nucleic acidmolecules produced by such methods and vectors and host cells comprisingsuch nucleic acid molecules. The invention further relates tocompositions for facilitating cloning of amplified nucleic acidmolecules.

2. Related Art

Cloning of Nucleic Acid Molecules

In examining the structure and physiology of an organism, tissue orcell, it is often desirable to determine its genetic content. Thegenetic framework of an organism is encoded in the double-strandedsequence of nucleotide bases in the deoxyribonucleic acid (DNA) which iscontained in the somatic and germ cells of the organism. The geneticcontent of a particular segment of DNA, or gene, is only manifested uponproduction of the protein which the gene encodes. In order to produce aprotein, a complementary copy of one strand of the DNA double helix (the“coding” strand) is produced by polymerase enzymes, resulting in aspecific sequence of ribonucleic acid (RNA). This particular type ofRNA, since it contains the genetic message from the DNA for productionof a protein, is called messenger RNA (mRNA).

Within a given cell, tissue or organism, there exist myriad mRNAspecies, each encoding a separate and specific protein. This factprovides a powerful tool to investigators interested in studying geneticexpression in a tissue or cell—mRNA molecules may be isolated andfurther manipulated by various molecular biological techniques, therebyallowing the elucidation of the full functional genetic content of acell, tissue or organism.

One common approach to the study of gene expression is the production ofcomplementary DNA (cDNA) clones. In this technique, the mRNA moleculesfrom an organism are isolated from an extract of the cells or tissues ofthe organism. This isolation often employs solid chromatographymatrices, such as cellulose or agarose, to which oligomers of thymidine(T) have been complexed. Since the 3′ termini on most eukaryotic mRNAmolecules contain a string of adenosine (A) bases, and since A binds toT, the mRNA molecules can be rapidly purified from other molecules andsubstances in the tissue or cell extract. From these purified mRNAmolecules, cDNA copies may be made using one or more polypeptides havingreverse transcriptase (RT) activity, which results in the production ofsingle-stranded cDNA molecules. The single-stranded cDNAs may then beconverted into a complete double-stranded DNA copy (i.e., adouble-stranded cDNA) of the original mRNA (and thus of the originaldouble-stranded DNA sequence, encoding this mRNA, contained in thegenome of the organism) by the action of a polypeptide having nucleicacid polymerase activity, such as a DNA polymerase. The protein-specificdouble-stranded cDNAs can then be inserted into a plasmid or viralvector (also called cloning vehicles), using controlled restrictionenzyme digestion and ligation of the cDNA and the vehicle. The resultingcDNA-vehicle construct is then introduced into a bacterial host, yeast,animal or plant cell and the host cells are then grown in culture media,resulting in a population of host cells containing (or in some cases,expressing) the gene of interest.

This entire process, from isolation of mRNA to insertion of the cDNAinto a plasmid or vector to growth of host cell populations containingthe isolated gene, is termed “cDNA cloning.” If cDNAs are prepared froma number of different mRNAs, the resulting set of cDNAs is called a“cDNA library” which represents a population of genes comprising thefunctional genetic information present in the source cell, tissue ororganism.

A variety of procedures are useful to clone genes. One such methodentails analyzing a library of cDNA inserts (derived from a cellexpressing the corresponding protein) for the presence of an insertwhich contains the desired gene. Such an analysis may be conducted bytransfecting cells with the vector, inducing the expression of theprotein, and then assaying for protein expression, for example, byimmunoreaction with an antibody which is specific for the desiredprotein.

Alternatively, in order to detect the presence of the desired gene, onemay employ an oligonucleotide (or set of oligonucleotides) which have anucleotide sequence that is complementary to the oligonucleotidesequence or set of sequences that codes for the desired protein. Sucholigonucleotides are used to detect and/or isolate the desired gene byselective hybridization. Techniques of nucleic acid hybridization aredisclosed by Maniatis, T., et al., In: Molecular Cloning, a LaboratoryManual, Cold Spring Harbor, N.Y. (1982), and by Haymes, B. D., et al.,In: Nucleic Acid Hybridization, a Practical Approach, IRL Press,Washington, D.C. (1985), which references are herein incorporated byreference.

In addition to the above methods, most commonly used cloning vectorshave an indicator gene which results in the expression of a specificphenotype in host cells containing the vector (e.g., blue colonies forhost cells containing vectors that carry lacZα; see Maniatis, T., etal., Id.). Insertion of heterologous nucleic acid sequences intomultiple cloning sites in such vectors interrupts or inactivates theindicator gene, resulting in non-expression of the phenotype (e.g.,white colonies for the above-described host cells containing lacZαvectors). Such an approach provides a convenient means fordifferentiating recombinant clones (i.e., those forming white colonies)from non-recombinant clones (i.e., those forming blue colonies).However, this approach does not prevent the growth of non-recombinantclones.

Nucleic Acid Amplification

Soon after their identification and characterization, it was recognizedthat the activities of the various enzymes and cofactors involved innucleic acid synthesis could be exploited in vitro to dramaticallyincrease the concentration of, or “amplify,” one or more selectednucleotide sequences. For many medical, diagnostic and forensicapplications, amplification of a particular nucleic acid molecule isessential to allow its detection in, or isolation from, a sample inwhich it is present in very low amounts. More recently, in vitroamplification of specific genes has provided powerful and less costlymeans to facilitate the production of therapeutic proteins by molecularbiological techniques, and may have applications in genetic therapy aswell.

While a variety of nucleic acid amplification processes have beendescribed, the most commonly employed is the Polymerase Chain Reaction(PCR) technique disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. Inthis process, a sample containing the nucleic acid sequence to beamplified (the “target sequence”) is first heated to denature orseparate the two strands of the nucleic acid. The sample is then cooledand mixed with specific oligonucleotide primers which hybridize to thetarget sequence. Following this hybridization, a buffered aqueoussolution containing at least one polypeptide having DNA polymeraseactivity is added to the sample, along with a mixture of the dNTPs thatare linked by the polymerase to the replicating nucleic acid strand.After allowing polymerization to proceed to completion, the products areagain heat-denatured, subjected to another round of primer hybridizationand polymerase replication, and this process is repeated any number oftimes. Since each nucleic acid product of a given cycle of this processserves as a template for production of two new nucleic acid molecules(one from each parent strand), the PCR process results in an exponentialincrease in the concentration of the target sequence. Thus, in awell-controlled, high-fidelity PCR process, as few as 20 cycles canresult in an over one million-fold amplification of the target nucleicacid sequence (See U.S. Pat. Nos. 4,683,195 and 4,683,202).

Other techniques for amplification of target nucleic acid sequences havealso been developed. For example, Walker et al. (U.S. Pat. No.5,455,166; EP 0 684 315) described a method called Strand DisplacementAmplification (SDA), which differs from PCR in that it operates at asingle temperature and uses a polymerase/endonuclease combination ofenzymes to generate single-stranded fragments of the target DNAsequence, which then serve as templates for the production ofcomplementary DNA (cDNA) strands. An alternative amplificationprocedure, termed Nucleic Acid Sequence-Based Amplification (NASBA) wasdisclosed by Davey et al. (U.S. Pat. No. 5,409,818; EP 0 329 822).Similar to SDA, NASBA employs an isothermal reaction, but is based onthe use of RNA primers for amplification rather than DNA primers as inPCR or SDA.

Amplification-Based Cloning

Standard cloning techniques such as those described above are oftenuseful for cloning nucleic acid sequences that are expressed atrelatively high levels in the source cells or tissues. However, thesetechniques frequently are not particularly sensitive when the startingsamples contain only low levels of the nucleic acid molecule ofinterest. This problem is particularly important when the tissue or cellsamples are themselves present in low quantities (as in many medical orforensic applications), or when the specific nucleotide sequence ispresent or expressed at low levels in the cell/tissue samples.

Amplification-based cloning of nucleic acid molecules, particularly thatemploying PCR, has been used in the attempt to overcome the lack ofsensitivity of earlier approaches (see, e.g., Lee, C. C., et al.,Science 239:1288-1291 (1988)). There are a number of methods availablefor performing such cloning.

In one such method, restriction enzyme sites can be incorporated intothe PCR primers; the PCR-generated nucleic acid molecules will thuscontain these restriction sites. For cloning of these specificsequences, these amplified nucleic acid molecules can then be digestedwith restriction enzymes, the digested fragments ligated into anappropriate site within a plasmid vector, and the vector incorporatedinto a host cell.

Alternatively, PCR products generated by Taq DNA polymerase, whichtypically contain an additional deoxyadenosine (dA) residue at their 3′termini, can be cloned into specific cloning vectors containing 3′deoxythymidine (dT) overhangs which provide a specific recognitionsequence for the 3′ A residue on the PCR product. This process, oftenreferred to as “TA cloning,” provides a means of directly cloningPCR-amplified nucleic acid molecules without the need for preparation ofprimers with specific restriction sites (see U.S. Pat. No. 5,487,993,which is incorporated herein by reference in its entirety).

In other cloning methods, blunt-end PCR fragments generated by cleavagewith certain restriction enzymes (e.g., SmaI, SspI or ScaI) can becloned into blunt-end insertion sites of cloning vectors (see, e.g.,Ausubel, F. M., et al., eds., “Current Protocols in Molecular Biology,”New York: John Wiley & Sons, Inc., pp. 3.16.1-3.16.11 (1995)), orPCR-amplified nucleic acid molecules can be cloned using uracil DNAglycosylase (UDG; see U.S. Pat. No. 5,137,814, which is incorporatedherein by reference in its entirety). Such blunt-end cloning may also befacilitated by treatment of Taq-amplified PCR products, which contain dAoverhangs as described above, with T4 DNA polymerase to remove the dAoverhangs (a procedure often termed “polishing”) followed by insertionof the resulting blunt-end fragments into blunt-end vector insertionsites as generally described above.

However, the cloning of amplified nucleic acid molecules, especially byrestriction enzyme digestion and insertion into cloning vehicles, isusually not simple and straightforward. Problems that plague theinvestigator are low cloning efficiencies (i.e., a low number ofrecombinant clones obtained per transformation) and cloning artifacts(i.e., recombinant clones which contain a modified insert). The probablecause of such technical limitations is residual polymerase activitywhich remains in the reaction mixture after the amplification process(see Bennet, B. L., and Molenaar, A. J., BioTechniques 16:36-37 (1994)).In fact, it has been shown that after 30 rounds of amplification understandard PCR conditions, sufficient residual polymerase activity ispresent in the reaction mixture to conduct an additional 30 rounds ofamplification. Upon digestion of the termini of the amplified nucleicacid molecules with restriction endonucleases to generate 3′ recessed(“sticky”) ends in the initial stages of cloning, this residualpolymerase can utilize remaining dNTPs in the sample to fill in the 3′ends to regenerate an undesirable blunt end. This interference resultsin poor ligation of the digested insert into a prepared recipientcloning vector which has been manipulated to possess recessed endscompatible with those of the insert. In fact, even the addition of asingle nucleotide to the 3′ sticky end can inhibit the ligation processand increase the number of incorrect recombinants that an operator mustscreen. An additional complication is that if the insert is to beligated into an expression vector for transformation into a host cell toultimately generate a protein encoded by the insert, the addition ofnucleotides to the digested amplification products can often shift thereading frame of the insert and result in expression of an incomplete,mutant and/or nonfunctional protein, especially if the promoter residesin the cloning vector 5′ to the insert.

One often-used approach to attempting to solve this technical probleminvolves multiple organic phenol/chloroform extractions of the amplifiednucleic acid molecules, prior to cloning, to aid in the removal of theresidual polymerases. Analogous methods involve similar time-consumingtechnical manipulations such as successive rounds of ethanolprecipitation and agarose gel purification. While such techniques mayreduce the content of the amplifying polymerase to some extent, theyalso usually result in reduced yields of clonable product due to loss,destruction and/or structural alteration of the amplified nucleic acidmolecules during purification. Thus, the temporal and economicconstraints to efficient and high-yield cloning ofamplified nucleic acidmolecules have yet to be overcome.

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to methods that overcome thesetemporal and economic constraints, providing for high-efficiency andrapid cloning of nucleic acid molecules, in particular amplified nucleicacid molecules. Specifically, the methods of the invention entail theuse of one or more inhibitors of polymerases in the cloning procedure,whereby residual polymerase activity remaining in the reaction mixtureafter nucleic acid synthesis or amplification is inactivated orinhibited, such that the nucleic acid molecules may be efficientlyligated into a cloning vector.

In one embodiment, the cloning methods of the invention comprise (a)amplifying or synthesizing one more nucleic acid molecules in thepresence of one of more polypeptides having polymerase activity toproduce copies of the nucleic acid molecules; and (b) incubating theamplified or synthesized nucleic acid molecules with one or moreinhibitors of the polypeptides having polymerase activity underconditions sufficient to inhibit or inactivate the polymerase activity.These methods of the invention may further comprise digesting theamplified or synthesized nucleic acid molecules with one or morerestriction endonucleases, to produce digested nucleic acid molecules,ligating the amplified, synthesized or digested nucleic acid moleculesinto one or more vectors to form one or more genetic constructs, andtransforming the genetic constructs into one or more host cells.Preferably, the inhibition or inactivation of the polypeptides havingpolymerase activity increases the efficiency of cloning of theamplified, synthesized or digested nucleic acid molecules into one ormore vectors. In addition, the inhibitors used in these methodspreferably prevent or inhibit modification of one or more termini of theamplified or digested nucleic acid molecules, and allow increasedefficiency ofcloning ofthe amplified, synthesized or digested nucleicacid molecules into one or more vectors.

The invention also relates to nucleic acid molecules produced by theabove-described methods.

According to the invention, the amplification step of theabove-described methods may comprise:

-   -   (a) contacting a first nucleic acid molecule, a first primer        nucleic acid molecule which is complementary to a portion of the        first nucleic acid molecule, a second nucleic acid molecule and        a second primer nucleic acid molecule which is complementary to        a portion of the second nucleic acid molecule, with one or more        polypeptides having polymerase activity;    -   (b) incubating the molecules under conditions sufficient to form        a third nucleic acid molecule complementary to all or a portion        of the first nucleic acid molecule and a fourth nucleic acid        molecule complementary to all or a portion of the second nucleic        acid molecule;    -   (c) denaturing the first and third and the second and fourth        nucleic acid molecules; and    -   (d) repeating steps (a) through (c) one or more times.

In another aspect, the invention relates to such methods wherein thefirst and/or second primer nucleic acid molecules comprise one or morerecombination sites (recombinase recognition sites) or portions thereof.Nucleic acid molecules synthesized according to this aspect of theinvention thus will comprise one or more recombination sites or portionsthereof, thereby facilitating easy movement or exchange of nucleic acidsegments between different synthesized nucleic acid molecules using oneor more recombinase proteins, as described below.

In accordance with the invention, the nucleic acid synthesis step in theabove methods may comprise:

-   -   (a) mixing a nucleic acid template (e.g., and RNA or a DNA        molecule, preferably an mRNA molecule) with one or more        polypeptides having polymerase activity; and    -   (b) incubating the mixture under conditions sufficient to make a        nucleic acid molecule complementary to all or a portion of the        template.

In preferred such aspects, the one or more DNA molecules synthesized bythe above methods may be one or more double-stranded cDNA molecules.

The polypeptides having polymerase activity that are preferred for usein these methods of the invention maybe DNA polymerases (includingthermostable DNA polymerases) or reverse transcriptases. Preferred DNApolymerases include Taq DNA polymerase, Tne DNA polymerase, Tma DNApolymerase, Pfu DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase,Pwo DNA polymerase, Bst DNA polymerase, Bca DNA polymerase, VENT™ DNApolymerase, DEEPVENT™ DNA polymerase, T7 DNA polymerase, DNA polymeraseIII, Klenow fragment DNA polymerase, Stoffel fragment DNA polymerase,and mutants, fragments or derivatives thereof. Preferred reversetranscriptases include M-MLV reverse transcriptase, RSV reversetranscriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAVreverse transcriptase, HIV reverse transcriptase, M-MLV H⁻ reversetranscriptase, RSV H⁻ reverse transcriptase, AMV H⁻ reversetranscriptase, RAV H⁻ reverse transcriptase, MAV H⁻ reversetranscriptase and HIV H⁻ reverse transcriptase, and mutants, fragmentsor derivatives thereof.

Preferred polymerase inhibitors for use in the methods of the presentinvention include, but are not limited to, antibodies (particularlyanti-Taq, anti-Tne, anti-Pfu or anti-Tma antibodies) or fragmentsthereof, chemical compounds, antibiotics, heavy metals, acids, metalchelators, nucleotide analogues, sulfhydryl reagents, anionicdetergents, polyanions, captan((N-[trichloromethyl]-thio)-4-cyclohexene-1,2-dicarboximide), acidicpolysaccharides, and combinations thereof.

In another aspect, the invention relates to methods of ligating anamplified, synthesized or digested nucleic acid molecule into a vectorwith increased efficiency, comprising:

-   -   (a) forming a mixture comprising the nucleic acid molecule and        one or more polymerase inhibitors; and    -   (b) ligating the nucleic acid molecule into one or more vectors        to form one or more genetic constructs.

The mixtures used in these methods may optionally further comprise oneor more polypeptides having polymerase activity. In addition, thesemethods of the invention may further comprise transforming the one ormore genetic constructs into one or more host cells.

The invention also relates to methods for cloning one or more nucleicacid molecules into one or more vectors, comprising:

-   -   (a) forming a mixture comprising the nucleic acid molecules to        be cloned (which may be amplified, synthesized or digested        nucleic acid molecules), the vectors and one or more polymerase        inhibitors; and    -   (b) ligating the nucleic acid molecules into the vectors to form        one or more genetic constructs.

In another embodiment, the invention relates to such cloning methodswherein the one or more nucleic acid molecules and/or one or morevectors may comprise one or more engineered recombination sites.

In preferred such methods, the nucleic acid molecules are cDNAmolecules.

These methods of the invention may further comprise transforming the oneor more genetic constructs into one or more host cells.

In another embodiment, the invention relates to methods for cloning oneor more nucleic acid molecules into one or more vectors, comprising:

-   -   (a) forming a mixture comprising the nucleic acid molecules to        be cloned (which may be synthesized or amplified nucleic acid        molecules), one or more polymerase inhibitors and one or more        restriction endonucleases; and    -   (b) ligating the nucleic acid molecules into one or more vectors        to form one or more genetic constructs.

In another embodiment, the invention relates to such cloning methodswherein the one or more nucleic acid molecules and/or one or morevectors may comprise one or more engineered recombination sites.

The mixtures used in these methods may optionally further comprise oneor more polypeptides having polymerase activity. In addition, thesemethods of the invention may further comprise transforming the one ormore genetic constructs into one or more host cells. In one preferredsuch method, the polymerase inhibitors and the restriction endonucleasesmay be added simultaneously. In another preferred method, the polymeraseinhibitors and the restriction endonucleases may be added sequentially.

Methods of the invention may involve any standard cloning methods inwhich a nucleic acid molecule is inserted into a vector. In particular,the invention concerns the use of topoisomerase, which can cleave avector to produce 3′ dT overhangs and ligate an amplified fragment whichcontains 3′ dA overhangs (produced, for example, by Taq DNA polymerase).Other enzymes for cleaving and ligating (e.g., DNA-modifying enzymes),used in cloning nucleic acid molecules into vectors, may also be used inaccordance with the invention. In the above-described methods whereinthe amplification primers and/or cloned nucleic acid molecules containone or more engineered recombination sites, for example, one or morerecombination proteins may be used in the above-noted standard cloningmethods. Recombination proteins which may be used in accordance withthis aspect of the invention include but are not limited tosite-specific recombinases, such as (a) the integrase family ofrecombinases (Argos et al. EMBO J. 5:433-440 (1986)) includingbacteriophage λ integrase, (Landy, A. (1993) Current Opinions inGenetics and Devel. 3:699-707), Cre from bacteriophage P1 (Hoess andAbremski (1990) In Nucleic Acids and Molecular Biology, vol. 4. Eds.:Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109),and FLP from Saccharomyces cerevisiae (Broach et al., Cell 29:227-234(1982)); and (b) the resolvase family of recombinases (e.g., γδ, Tn3resolvase, Hin, Gin, and Cin) (Maeser and Kahnmann (1991) Mol. Gen.Genet. 230:170-176). Other site-specific recombinases may also be usedin accordance with the methods of the invention, including thesite-specific recombination proteins encoded by bacteriophage lambda,phi 80, P22, P2, 186, P4 and P1 which are known in the art.

In another aspect, the invention also relates to kits for cloning anamplified, synthesized or digested nucleic acid molecule. Kits accordingto the invention may comprise one or more containers containing one ormore of the above-described polymerase inhibitors. Such kits may furthercomprise one or more additional containers containing, for example, oneor more polypeptides having polymerase activity, one or more primernucleic acid molecules, one or more nucleotides, one or morepolypeptides having reverse transcriptase activity, one or more ligases,one or more vectors, one or more host cells (which may be competent fortransformation), one or more topoisomerases and one or more restrictionendonucleases.

The invention also relates to compositions comprising one or morerestriction endonucleases and one or more of the above-describedpolymerase inhibitors, either or both of which may be stable uponstorage. Compositions of the invention also comprise the above-describedpolymerase inhibitors and one or more DNA modifying enzymes orcombinations thereof (such as ligases, kinases, phosphatases, nucleases,endonucleases, topoisomerases, gyrases, terminal deoxynucleotidyltransferases, etc.) Compositions according to this aspect of theinvention may further comprise one or more additional components,including, for example, one or more nucleic acid molecules or one ormore suitable buffers.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of the following drawings and descriptionof the invention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an autoradiograph of a 552 bp amplification product obtainedby Taq polymerase-mediated amplification of a 664 bp amplicon of the 3′end of the chloramphenicol acetyltransferase (CAT) gene. Followingamplification, the 552 bp product was incubated in the presence orabsence of two different preparations of anti-Taq antibodies prior tobeing digested with EcoRI and HindIII in the presence of ³²P-dATP, andsamples were then resolved on a 1% TBE-agarose gel followed byautoradiography. Lanes 1, 2, 6 and 7: no antibody treatment; lanes 3, 4and 5: antibody preparation #1, at 1 unit, 0.67 unit and 0.33 unit,respectively; lanes 8, 9 and 10: antibody preparation #2, at 1 unit,0.67 unit and 0.33 unit, respectively; lane 11: restriction enzymedigestion followed by treatment of samples with Klenow fragment(positive control).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms conventionally usedin the fields of molecular biology and protein engineering, andtherefore generally understood by those of routine skill in the art, areutilized extensively. Certain terms as used herein, however, havespecific meanings for the purposes of the present invention. In order toprovide a clear and consistent understanding of the specification andclaims, and the scope to be given such terms, the following definitionsare provided.

The term “polypeptide” is used herein to mean a sequence of contiguousamino acids, of any length. As used herein, the terms “peptide” or“protein” may be used interchangeably with the term “polypeptide.”

As used herein, “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes deoxyribonucleosidetriphosphates (“dNTPs”) such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, orderivatives thereof. Such derivatives include, for example, [αS]dATP,7-deaza-dGTP and 7-deaza-dATP. The term nucleotide as used herein alsorefers to dideoxyribonucleoside triphosphates (“ddNTPs”) and theirderivatives, including, but not limited to, ddATP, ddCTP, ddGTP, ddITP,and ddTTP. In addition, the term nucleotide includes ribonucleosidetriphosphates (rNTPs) such as rATP, rCTP, rITP, rUTP, rGTP, rTTP andtheir derivatives, which are analogous to the above-described dNTPs andddNTPs except that the rNTPs comprise ribose instead of deoxyribose ordideoxyribose in their sugar-phosphate backbone. According to thepresent invention, a “nucleotide” maybe unlabeled or detectably labeledby well known techniques. Detectable labels include, for example,radioactive isotopes, fluorescent labels, chemiluminescent labels,bioluminescent labels and enzyme labels.

The term “nucleic acid molecule” as used herein refers to a sequence ofcontiguous nucleotides (dNTPs or ddNTPs, or combinations thereof) whichmay encode a full-length polypeptide or a fragment of any lengththereof, or which may be non-coding.

The term “dNTP” (plural “dNTPs”) generically refers to thedeoxynucleoside triphosphates (e.g., dATP, dCTP, dGTP, dTTP, dUTP, dITP,7-deaza-dGTP, αdATP, αdTTP, αdGTP and αdCTP), and the term “ddNTP”(plural “ddNTPs”) to their dideoxy counterparts, that are incorporatedby polymerase enzymes into newly synthesized nucleic acids.

The term “unit” as used herein refers to the activity of an enzyme. Whenreferring to a DNA polymerase, one unit of activity is the amount ofenzyme that will incorporate 10 nanomoles of dNTPs into acid-insolublematerial (i.e., DNA or RNA) in 30 minutes under standard primed DNAsynthesis conditions.

The terms “stable” and “stability” as used herein generally mean theretention by an enzyme of at least 70%, preferably at least 80%, andmost preferably at least 90%, of the original enzymatic activity (inunits) after the enzyme or composition containing the enzyme has beenstored for at least four weeks at a temperature of about 20-25° C., atleast one year at a temperature of about 4° C. or at least 2 years at atemperature of −20° C.

As used herein, a “cloning vector” or “cloning vehicle” is a plasmid,cosmid or phage DNA or other DNA molecule which is able to replicateautonomously in a host cell, and which is characterized by one or asmall number of restriction endonuclease recognition sites at which suchDNA sequences may be cut in a determinable fashion without loss of anessential biological function of the vector, and into which DNA may bespliced in order to bring about its replication and cloning. The cloningvector or vehicle may further contain a marker suitable for use in theidentification of cells transformed with the cloning vector. Markers,for example, are tetracycline resistance or ampicillin resistance.

As used herein, a “primer” refers to a single-stranded oligonucleotidethat is extended by covalent bonding of nucleotide monomers duringamplification or polymerization of a DNA molecule

The term “template” as used herein refers to a double-stranded orsingle-stranded nucleic acid molecule which is to be amplified,synthesized or sequenced. In the case of a double-stranded DNA molecule,denaturation of its strands to form a first and a second strand isperformed before these molecules may be amplified, synthesized orsequenced. A primer, complementary to a portion of a DNA template ishybridized under appropriate conditions and the DNA polymerase of theinvention may then synthesize a DNA molecule complementary to saidtemplate or a portion thereof. The newly synthesized DNA molecule,according to the invention, maybe equal or shorter in length than theoriginal DNA template. Mismatch incorporation or strand slippage duringthe synthesis or extension of the newly synthesized DNA molecule mayresult in one or a number of mismatched base pairs. Thus, thesynthesized DNA molecule need not be exactly complementary to the DNAtemplate.

The term “incorporating” as used herein means becoming a part of a DNAmolecule or primer.

As used herein, “amplification” refers to any in vitro method forincreasing the number of copies of a nucleotide sequence with the use ofa polymerase. Nucleic acid amplification results in the incorporation ofnucleotides into a nucleic acid (e.g., DNA) molecule or primer therebyforming a new nucleic acid molecule complementary to the nucleic acidtemplate. The formed nucleic acid molecule and its template can be usedas templates to synthesize additional nucleic acid molecules. As usedherein, one amplification reaction may consist of many rounds of nucleicacid synthesis. Amplification reactions include, for example, polymerasechain reactions (PCR). One PCR reaction may consist of 5 to 100 “cycles”of denaturation and synthesis of a nucleic acid molecule.

An “oligonucleotide” as used herein refers to a synthetic or naturalmolecule comprising a covalently linked sequence of nucleotides whichare joined by a phosphodiester bond between the 3′ position of thepentose of one nucleotide and the 5′ position of the pentose of theadjacent nucleotide.

As used herein, “thermostable” refers to an enzyme (such as apolypeptide having nucleic acid polymerase or reverse transcriptaseactivity) which is resistant to inactivation by heat. DNA polymerasessynthesize the formation of a DNA molecule complementary to asingle-stranded DNA template by extending a primer in the 5′-to-3′direction. This activity for mesophilic DNA polymerases may beinactivated by heat treatment. For example, the activities of T5 and T7DNA polymerases are totally inactivated by exposing the enzymes to atemperature of 90° C. for 30 seconds. As used herein, a thermostable DNApolymerase activity is more resistant to heat inactivation than amesophilic DNA polymerase. However, a thermostable DNA polymerase doesnot mean to refer to an enzyme which is totally resistant to heatinactivation; thus heat treatment may reduce the DNA polymerase activityto some extent. A thermostable DNA polymerase typically will also have ahigher optimum temperature than mesophilic DNA polymerases.

The terms “hybridization” and “hybridizing” as used herein refer to thepairing of two complementary single-stranded nucleic acid molecules (RNAand/or DNA) to give a double-stranded molecule. As used herein, twonucleic acid molecules may be hybridized, although the base pairing isnot completely complementary. Accordingly, mismatched bases do notprevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used. In the presentinvention, the term “hybridization” refers particularly to hybridizationof an oligonucleotide to a DNA template molecule.

“Working concentration” is used herein to mean the concentration of areagent that is at or near the optimal concentration used in a solutionto perform a particular function (such as amplification or digestion ofa nucleic acid molecule). The working concentration of a reagent is alsodescribed equivalently as a “1× concentration” or a “1× solution” (ifthe reagent is in solution) of the reagent. Accordingly, higherconcentrations of the reagent may also be described based on the workingconcentration; for example, a “2× concentration” or a “2× solution” of areagent is defined as a concentration or solution that is twice as highas the working concentration of the reagent; a “5× concentration” or a“5× solution” is five times as high as the working concentration of thereagent; and so on.

The terms “recombinase” and “recombination protein” as used herein maybe used interchangeably, and refer to an excisive or integrativeprotein, enzyme, co-factor or associated protein that is involved inrecombination reactions involving one or more recombination sites, suchas an enzyme which catalyzes the exchange of DNA segments at specificrecombination sites. See, Landy, A., Ann. Rev. Biochem. 58:913-949(1989).

The terms “recognition sequence” or “recombination site” as used hereinrefer to a particular DNA sequence which a protein, DNA, or RNA molecule(e.g., a restriction endonuclease, a modification methylase, or arecombinase) recognizes and binds. For example, the recognition sequencefor Cre recombinase is loxP which is a 34 base pair sequence comprisedof two 13 base pair inverted repeats (serving as the recombinase bindingsites) flanking an 8 base pair core sequence. See FIG. 1 of Sauer, B.,Current Opinion in Biotechnology 5:521-527 (1994). Other examples ofrecognition sequences are the attB, attP, attL, and attR sequences whichare recognized by the recombinase enzyme λ Integrase. attB is anapproximately 25 base pair sequence containing two 9 base pair core-typeInt binding sites and a 7 base pair overlap region. attP is anapproximately 240 base pair sequence containing core-type Int bindingsites and arm-type Int binding sites as well as sites for auxiliaryproteins MF, FIS, and Xis. See Landy, Current Opinion in Biotechnology3:699-707 (1993). Such sites are also engineered according to thepresent invention to enhance methods and products.

The phrase “recombinational cloning” is used herein to mean a methodwhereby segments of DNA molecules are exchanged, inserted, replaced,substituted or modified, in vitro or in vivo.

Other terms used in the fields of recombinant DNA technology andmolecular and cell biology as used herein will be generally understoodby one of ordinary skill in the applicable arts.

Overview

The present invention is generally directed to methods that overcome theabove-described temporal and economic constraints that are typicallyencountered during attempts to clone amplified or synthesized nucleicacid molecules. Thus, the invention provides methods that result inhigh-efficiency and rapid cloning of amplified, synthesized or digestednucleic acid molecules. Specifically, the methods of the inventionentail the use of one or more inhibitors of nucleic acid polymerases inthe cloning procedure, whereby residual polymerase activity remaining inthe reaction mixture after amplification or synthesis is inactivated orinhibited. By the methods of the invention, amplified, synthesized ordigested nucleic acid molecules may be quickly and efficiently ligated(using ligases, topoisomerases, etc.) into cloning vectors, and thesevectors then inserted into host cells, for example for expression of thecloned nucleic acid molecules.

Methods according to this aspect of the invention may comprise one ormore steps. One example is a method of cloning an amplified orsynthesized nucleic acid molecule, comprising:

-   -   (a) amplifying or synthesizing one more nucleic acid molecules        in the presence of one or more polypeptides having polymerase        activity to produce amplified nucleic acid molecules; and    -   (b) incubating the nucleic acid molecules with one or more        inhibitors of the polypeptides having polymerase activity under        conditions sufficient to inhibit or inactivate the polymerase        activity.

Sources of Nucleic Acid Template Molecules

Using the methods of the invention, synthesized, amplified or digestednucleic acid molecules may be derived from a variety of sources. Nucleicacid molecules suitably cloned by the methods of the present inventionmay be DNA molecules (including cDNA molecules), RNA molecules(including polyadenylated RNA (polyA+RNA), messenger RNA (mRNA),transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules) or DNA-RNAhybrid molecules, and may be single-stranded or double-stranded.

The nucleic acid molecules to be cloned according to the methods of thepresent invention may be prepared synthetically according to standardorganic chemical synthesis methods that will be familiar to one ofordinary skill. More preferably, the nucleic acid molecules may beobtained from natural sources, such as a variety of cells, tissues,organs or organisms. Cells that may be used as sources of nucleic acidmolecules may be prokaryotic (bacterial cells, including those ofspecies of the genera Escherichia, Bacillus, Serratia, Salmonella,Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria,Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium,Helicobacter, Erwinia, Agrobacterium, Rhizobium, and Streptomyces) oreukaryotic (including fungi (especially yeasts), plants, protozoans andother parasites, and animals including insects (particularly Drosophilaspp. cells), nematodes (particularly Caenorhabditis elegans cells), andmammals (particularly human, rodent (rat or mice), monkey, ape, canine,feline, equine, bovine and ovine cells, and most particularly humancells)).

Mammalian somatic cells that may be used as sources of nucleic acidsinclude blood cells (reticulocytes and leukocytes), endothelial cells,epithelial cells, neuronal cells (from the central or peripheral nervoussystems), muscle cells (including myocytes and myoblasts from skeletal,smooth or cardiac muscle), connective tissue cells (includingfibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes andosteoblasts) and other stromal cells (e.g., macrophages, dendriticcells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes)may also be used as sources of nucleic acids for use in the invention,as may the progenitors, precursors and stem cells that give rise to theabove somatic and germ cells (e.g., embryonic stem cells). Also suitablefor use as nucleic acid sources are mammalian tissues or organs such asthose derived from brain, kidney, liver, pancreas, blood, bone marrow,muscle, nervous, skin, genitourinary, circulatory, lymphoid,gastrointestinal and connective tissue sources, as well as those derivedfrom a mammalian (including human) embryo or fetus.

Any of the above prokaryotic or eukaryotic cells, tissues and organs maybe normal, diseased, transformed, established, progenitors, precursors,fetal or embryonic. Diseased cells may, for example, include thoseinvolved in infectious diseases (caused by bacteria, fungi or yeast,viruses (including HIV) or parasites), in genetic or biochemicalpathologies (e.g., cystic fibrosis, hemophilia, Alzheimer's disease,muscular dystrophy or multiple sclerosis) or in cancerous processes.Transformed or established animal cell lines may include, for example,COS cells, CHO cells, VERO cells, BHK cells, HeLa cells, HepG2 cells,K562 cells, F9 cells and the like. Other cells, cell lines, tissues,organs and organisms suitable as sources ofnucleic acids for use in thepresent invention will be apparent to one of ordinary skill in the art.

In addition, such nucleic acid molecules and cDNA libraries may beobtained commercially, for example from Life Technologies, Inc.(Rockville, Md.) and other commercial suppliers that will be familiar tothe skilled artisan.

Once the starting cells, tissues, organs, libraries or other samples areobtained, nucleic acid molecules to be cloned by the methods of theinvention may be isolated by methods that are well-known in the art(See, e.g., Maniatis, T., et al., Cell 15:687-701 (1978); Okayama, H.,and Berg, P., Mol. Cell. Biol. 2:161-170 (1982); Gubler, U., andHoffinan, B. J., Gene 25:263-269 (1983)). The nucleic acid moleculesthus isolated may then be cloned using the methods of the presentinvention.

Amplified Nucleic Acid Molecules

Preferably, the nucleic acid molecules to be cloned are amplifiednucleic acid molecules. Nucleic acid molecules may be amplified by anumber of methods, which may comprise one or more steps. For example,one such method comprises (a) contacting a first nucleic acid molecule,a first primer molecule which is complementary to a portion of the firstnucleic acid molecule, a second nucleic acid molecule and a secondprimer molecule which is complementary to a portion of the secondnucleic acid molecule, with one or more polypeptides having polymeraseactivity; (b) incubating the molecules and one or more polypeptidesunder conditions sufficient to form a third nucleic acid moleculecomplementary to all or a portion of the first nucleic acid molecule anda fourth nucleic acid molecule complementary to all or a portion of thesecond nucleic acid molecule; (c) denaturing the first and third and thesecond and fourth nucleic acid molecules; and (d) repeating steps (a)through (c) one or more times. Such amplification methods may beaccomplished by any of a variety of techniques, including but notlimited to use of the polymerase chain reaction (PCR; U.S. Pat. Nos.4,683,195 and 4,683,202), Strand Displacement Amplification (SDA; U.S.Pat. No. 5,455,166), and Nucleic Acid Sequence-Based Amplification(NASBA; U.S. Pat. No. 5,409,818); particularly preferred is PCR.

In another aspect, the invention relates to the above-described nucleicacid synthesis or amplification methods, wherein the first and/or secondprimer nucleic acid molecules used in the above-described amplificationmethods comprise one or more recombination sites (recombinaserecognition sites) or portions thereof. Nucleic acid moleculessynthesized or amplified according to this aspect of the invention thuswill comprise one or more recombination sites or portions thereof,thereby facilitating easy movement or exchange of nucleic acid segmentsbetween different synthesized nucleic acid molecules using one or morerecombinase proteins in a process termed recombinational cloning, asdescribed below. Preferred combinations of recombinationsites/recombination proteins for use according to this aspect of theinvention include the Integrase/att system from bacteriophage λ (Landy,A. (1993) Current Opinions in Genetics and Devel. 3:699-707); theCre/loxP system from bacteriophage P1 (Hoess and Abremski (1990) InNucleic Acids and Molecular Biology, vol. 4. Eds.: Eckstein and Lilley,Berlin-Heidelberg: Springer-Verlag; pp. 90-109); the FLP/FRT system fromthe Saccharomyces cerevisiae 2μ circle plasmid (Broach et al. Cell29:227-234 (1982); the resolvase family (e.g., γδ, Tn3 resolvase, Hin,Gin, and Cin) (Maeser and Kahnmann (1991) Mol. Gen. Genet. 230:170-176);and site-specific recombination proteins encoded by bacteriophagelambda, phi 80, P22, P2, 186, P4 and P1. Methods for preparation ofprimers comprising one or more recombination sites, and use of suchprimers in synthesizing or amplifying one or more nucleic acid moleculeproducts which comprise one or more recombination sites, are describedin detail in commonly owned, co-pending U.S. application Ser. No.08/486,139, filed Jun. 7, 1995; 60/065,930, filed Oct. 24, 1997; andSer. No. 09/005,476, filed Jan. 12, 1998, the disclosures of all ofwhich are incorporated herein by reference in their entireties.

In one embodiment of the present invention, the amplified nucleic acidfragments may be cloned (ligated) directly into one or more vectors toproduce one or more genetic constructs. The genetic constructs may thenbe transformed into one or more host cells.

In other cloning methods, amplified molecules cleaved or digested withone or more restriction enzymes or one or more recombination proteins asdescribed in more detail below can be cloned into appropriate insertionsites of cloning vectors (see, e.g., Ausubel, F. M., et al., eds.,“Current Protocols in Molecular Biology,” New York: John Wiley & Sons,Inc., pp. 3.16.1-3.16.11 (1995)). Restriction enzymes used for cleavageof the amplified molecules may include blunt-end cutters (e.g., SmaI,SspI, ScaI, etc.) and sticky-end cutters (e.g., HindIII, BamHI, KpnI,etc.). Amplified nucleic acid molecules can also be cloned using uracilDNA glycosylase (UDG; see U.S. Pat. No. 5,137,814, which is incorporatedherein by reference in its entirety).

In another aspect of the invention, restriction enzyme sites can beincorporated into the amplification primers; the amplified nucleic acidmolecules will thus contain these restriction sites. For cloning ofthese specific sequences, these amplified nucleic acid molecules canthen be digested with restriction enzymes, the digested fragmentsligated into an appropriate site within a plasmid vector, and the vectorincorporated into a host cell as described in more detail below.

In another aspect of the invention, recombination or recombinaserecognition sites can be incorporated into the amplification primers;the amplified nucleic acid molecules will thus contain theserecombination or recombinase recognition sites. These amplified nucleicacid molecules can then be treated with one or more recombinationsproteins as described below, to facilitate exchange or recombination ofone or more nucleic acid segments between different amplified nucleicacid molecules, in a process known as recombinational cloning. Theresulting recombined nucleic acid molecules may then be inserted into aplasmid vector, and the vector incorporated into a host cell asdescribed in more detail below.

Amplified products generated by DNA polymerases which incorporate anadditional deoxyadenosine (dA) residue at the 3′ termini of the products(e.g., Taq DNA polymerase), can be cloned into specific cloning vectorscontaining 3′ deoxythymidine (dT) overhangs which provide a specificrecognition sequence for the 3′ A residue on the amplified product. Thisprocess, often referred to as “TA cloning,” provides a means of directlycloning amplified nucleic acid molecules without the need forpreparation of primers with specific restriction sites (see U.S. Pat.No. 5,487,993, which is incorporated herein by reference in itsentirety). Alternatively, a ligase-independent strategy for cloning maybe used (such as Topo-TA Cloning®; Invitrogen, Carlsbad, Calif.).Blunt-end cloning of such amplified molecules containing dA overhangsmay be facilitated by using T4 DNA polymerase to remove the dA overhangs(a procedure often termed “polishing”) followed by insertion of theresulting blunt-end fragments into blunt-end vector insertion sites asdescribed in more detail below.

Polymerases and Reverse Transcriptases

A variety of polypeptides having polymerase activity are useful in themethods of the present invention. Included among these polypeptides areenzymes such as nucleic acid polymerases (including DNA polymerases andRNA polymerases), as well as polypeptides having reverse transcriptase(i.e., RNA-dependent DNA polymerase) activity.

Polypeptides having reverse transcriptase activity that may beadvantageously used in the present methods include, but are not limitedto, Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, RousSarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus(AMV) reverse transcriptase, Rous-Associated Virus (RAV) reversetranscriptase, Myeloblastosis Associated Virus (MAV) reversetranscriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase,retroviral reverse transcriptase, retrotransposon reverse transcriptase,hepatitis B reverse transcriptase, cauliflower mosaic virus reversetranscriptase, bacterial reverse transcriptase, Thermus thermophilus(Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoganeopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNApolymerase, Thermococcus litoralis (Tli or VENT™) DNA polymerase,Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT™ DNA polymerase,Pyrococcus woosii (Pwo) DNA polymerase, Bacillus sterothermophilus (Bst)DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobusacidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNApolymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermus ruber (Tru)DNA polymerase, Thermus brockianus (DYNAZYME™) DNA polymerase,Methanobacterium thermoautotrophicum (Mth) DNA polymerase, and mutants,variants and derivatives thereof. Particularly preferred for use in theinvention are the variants of these enzymes that are substantiallyreduced in RNase H activity (i.e., “RNase H⁻” enzymes). By an enzyme“substantially reduced in RNase H activity” is meant that the enzyme hasless than about 20%, more preferably less than about 15%, 10% or 5%, andmost preferably less than about 2%, of the RNase H activity of awildtype or “RNase H⁺” enzyme such as wildtype M-MLV or AMV reversetranscriptases. The RNase H activity of any enzyme may be determined bya variety of assays, such as those described, for example, in U.S. Pat.No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res. 16:265(1988) and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), thedisclosures of all of which are fully incorporated herein by reference.Particularly preferred RNase H⁻ reverse transcriptase enzymes for use inthe invention include, but are not limited to, M-MLV H⁻ reversetranscriptase, RSV H⁻ reverse transcriptase, AMV H⁻ reversetranscriptase, RAV H⁻ reverse transcriptase, MAV H⁻ reversetranscriptase and HIV H⁻ reverse transcriptase. It will be understood byone of ordinary skill, however, that any enzyme capable of producing aDNA molecule from a ribonucleic acid molecule (i.e., having reversetranscriptase activity) that is substantially reduced in RNase Hactivity may be equivalently used in the compositions, methods and kitsof the invention.

Enzymes used in the invention may have distinct reverse transcriptionpause sites with respect to the template nucleic acid. Whether or nottwo enzymes have distinct reverse transcription pause sites may bedetermined by a variety of assays, including, for example,electrophoretic analysis of the chain lengths of DNA molecules producedby the two enzymes (Weaver, D. T., and DePamphilis, M. L., J. Biol.Chem. 257(4):2075-2086 (1982); Abbots, J., et al., J. Biol. Chem.268(14):10312-10323 (1993)), or by other assays that will be familiar toone of ordinary skill in the art. As described above, these distincttranscription pause sites may represent secondary structural andsequence barriers in the nucleic acid template which occur frequently athomopolymer stretches. Thus, for example, the second enzyme may reversetranscribe to a point (e.g., a hairpin) on the template nucleic acidthat is proximal or distal (i.e., 3′ or 5′) to the point to which thefirst enzyme reverse transcribes the template nucleic acid. Thiscombination of two or more enzymes having distinct reverse transcriptionpause sites facilitates production of full-length cDNA molecules sincethe secondary structural and sequence barriers may be overcome.

Polypeptides having reverse transcriptase activity for use in theinvention may be obtained commercially, for example from LifeTechnologies, Inc. (Rockville, Md.), Pharmacia (Piscataway, N.J.), Sigma(Saint Louis, Mo.) or Boehringer Mannheim Biochemicals (Indianapolis,Ind.). Alternatively, polypeptides having reverse transcriptase activitymay be isolated from their natural viral or bacterial sources accordingto standard procedures for isolating and purifying natural proteins thatare well-known to one of ordinary skill in the art (see, e.g., Houts, G.E., et al., J. Virol. 29:517 (1979)). In addition, the polypeptideshaving reverse transcriptase activity may be prepared by recombinant DNAtechniques that are familiar to one of ordinary skill in the art (see,e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis,D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376(1988)).

Nucleic acid polymerases such as DNA polymerases for use in the presentmethods may be isolated from natural or recombinant sources, bytechniques that are well-known in the art (See WO 92/06200, U.S. Pat.Nos. 5,455,170 and 5,466,591, WO 96/10640 and U.S. patent applicationSer. No. 08/370,190, filed Jan. 9, 1995), from a variety of thermophilicbacteria that are available commercially (for example, from AmericanType Culture Collection, Rockville, Md.) or may be obtained byrecombinant DNA techniques (see, e.g., WO 96/10640 and U.S. patentapplication Ser. No. 08/370,190, filed Jan. 9, 1995). Suitable for useas sources of thermostable polymerases or the genes thereof forexpression in recombinant systems are the thermophilic bacteria Thermusthermophilus, Thermococcus litoralis, Pyrococcus furiosus, Pyrococcuswoosii and other species of the Pyrococcus genus, Bacillussterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum,Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoganeapolitana, Thermotoga maritima and other species of the Thermotogagenus, and Methanobacterium thermoautotrophicum, and mutants, variantsor derivatives thereof. It is to be understood, however, thatthermostable DNA polymerases from other organisms may also be used inthe present invention without departing from the scope or preferredembodiments thereof. As an alternative to isolation, thermostable DNApolymerases are available commercially from, for example, LifeTechnologies, Inc. (Rockville, Md.), New England BioLabs (Beverly,Mass.), Finnzymes Oy (Espoo, Finland), Stratagene (La Jolla, Calif.),Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and Perkin ElmerCetus (Norwalk, Conn.).

DNA polymerases used in accordance with the invention may be any enzymethat can synthesize a DNA molecule from a nucleic acid template,typically in the 5′ to 3′ direction. The nucleic acid polymerases usedin the present invention may be mesophilic or thermophilic, and arepreferably thermophilic. Preferred mesophilic DNA polymerases include T7DNA polymerase, T5 DNA polymerase, Klenow fragment DNA polymerase, DNApolymerase III and the like. Preferred thermostable DNA polymerases thatmay be used in the methods of the invention include Taq, Tne, Tma, Pfu,Tfl, Tth, Stoffel fragment, VENT™ and DEEPVENT™ DNA polymerases, andmutants, variants and derivatives thereof (U.S. Pat. No. 5,436,149; U.S.Pat. No. 5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; Barnes, W.M., Gene 112:29-35 (1992); Lawyer, F. C., et al., PCR Meth. Appl.2:275-287 (1993); Flaman, J.-M., et al., Nucl. Acids Res.22(15):3259-3260 (1994)). For amplification of long nucleic acidmolecules (e.g., nucleic acid molecules longer than about 3-5 Kb inlength), at least two DNA polymerases (one substantially lacking 3′exonuclease activity and the other having 3′ exonuclease activity) aretypically used. See U.S. Pat. No. 5,436,149; U.S. Pat. No. 5,512,462;Barnes, W. M., Gene 112:29-35 (1992); and co-pending U.S. patentapplication Ser. No. 08/689,815, filed Feb. 14, 1997, the disclosures ofwhich are incorporated herein in their entireties. Examples of DNApolymerases substantially lacking in 3′ exonuclease activity include,but are not limited to, Taq, Tne(exo⁻), Tma(exo⁻), Pfu(exo⁻) Pwo(exo⁻)and Tth DNA polymerases, and mutants, variants and derivatives thereof.Nonlimiting examples of DNA polymerases having 3′ exonuclease activityinclude Pfu, DEEPVENT™, Tli/VENT™, Tne, Tma, and mutants, variants andderivatives thereof.

Polypeptides having nucleic acid polymerase and/or reverse transcriptaseactivity are preferably used in the present methods at a finalconcentration in solution of about 0.1-200 units per milliliter, about0.1-50 units per milliliter, about 0.1-40 units per milliliter, about0.1-36 units per milliliter, about 0.1-34 units per milliliter, about0.1-32 units per milliliter, about 0.1-30 units per milliliter, or about0.1-20 units per milliliter, and most preferably at a concentration ofabout 20 units per milliliter. Of course, other suitable concentrationsof reverse transcriptase enzymes and nucleic acid polymerases suitablefor use in the invention will be apparent to one of ordinary skill inthe art.

Cloning of Nucleic Acid Molecules

The methods of the invention may further comprise one or more additionalsteps designed to facilitate the cloning of the amplified or synthesizednucleic acid molecules. For example, nucleic acid molecules amplified orsynthesized as described above may be digested with one or morerestriction endonucleases, to produce a collection of digested nucleicacid molecules. Suitable methods and enzymes for use in digestingnucleic acid molecules will be familiar to one of ordinary skill in theart (see, e.g., Sambrook, J., et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress (1989)). Restriction endonucleases that may be advantageously usedin the methods of the invention include, but are not limited to, AluI,Eco47 III, EcoRV, FspI, HpaI, MscI, NruI, PvuII, RsaI, ScaI, SmaI, SspI,StuI, ThaI, AvaI, BamHI, BanII, BglII, ClaI, EcoRI, HindIII, HpaII,KpnI, MseI, NcoI, NdeI, NotI, PstI, PvuI, SacI/SstI, SalI, XbaI, XhoIand I-CeuI. Such restriction endonucleases are available commercially,for example from Life Technologies, Inc. (Rockville, Md.), Sigma (St.Louis, Mo.) and New England BioLabs (Beverly, Mass.). Topoisomerases orother nucleic acid-modifying enzymes may also be used.

In alternative cloning methods of the invention, amplified nucleic acidmolecules that comprise one or more recombination sites may be treatedwith one or more recombination proteins which recognize, bind to, andcleave the nucleic acid molecules at the specific recombination sites.Preferred recombination proteins for use in this aspect of the inventioninclude those described above, such as the Int, IHF or Xis integrases;Cre; γδ, Tn3 resolvase, Hin, Gin, Cin, Flp; and other recombinationproteins encoded by bacteriophage λ, phi 80, P22, P2, 186, P4 and P1.Appropriate methods using such recombination proteins in cloningofnucleic acid molecules comprising one or more recombination sites aredescribed in detail in commonly owned, co-pending U.S. application Ser.No. 08/486,139, filed Jun. 7, 1995; 60/065,930, filed Oct. 24, 1997; andSer. No. 09/005,476, filed Jan. 12, 1998, the disclosures of all ofwhich are incorporated herein by reference in their entireties.

Once the synthesized or amplified nucleic acid molecules have beendigested with one or more restriction enzymes or cleaved with one ormore recombination proteins, the digested or cleaved nucleic acidmolecules may be inserted (typically by ligation, for example using apolypeptide having nucleic acid ligase activity such as T4 DNA ligase,topoisomerase or the like) into one or more vectors, such as one or moreexpression vectors, to yield one or more genetic constructs.Alternatively, the amplified or synthesized nucleic acid molecules maybe ligated directly into one or more vectors without being digested orcleaved, to form one or more genetic constructs. Genetic constructsaccording to this aspect of the invention thus typically comprise theamplified, synthesized or digested/cleaved nucleic acid molecule (orfragments thereof) and the vector or cloning vehicle. These geneticconstructs may, in turn, be introduced into host cells using well-knowntechniques such as infection, transduction, transfection,electroporation and transformation, for the large-scale production ofcDNA libraries or plasmids comprising the amplified, synthesized ordigested/cleaved nucleic acid molecules, or for the expression of theamplified, synthesized or digested/cleaved nucleic acid molecules. Thevectors may be, for example, a phage, plasmid, viral or retroviralvector, and is preferably an expression vector as described below.Retroviral vectors may be replication-competent orreplication-defective. In the latter case, viral propagation generallywill occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced into mammalian or avian cells in a precipitate, such as acalcium phosphate precipitate, or in a complex with a charged lipid(e.g., LIPOFECTAMINE™; Life Technologies, Inc.; Rockville, Md.) or in acomplex with a virus (such as an adenovirus; see U.S. Pat. Nos.5,547,932 and 5,521,291) or components of a virus (such as viral capsidpeptides). If the vector is a virus, it may be packaged in vitro usingan appropriate packaging cell line and then transduced into host cells.

Preferred are vectors comprising cis-acting control regions to thenucleic acid molecule of interest. Appropriate trans-acting factors maybe supplied by the host, by a complementing vector or by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors may providefor specific expression of the amplified, synthesized ordigested/cleaved nucleic acid molecules, which may be inducible and/orcell type-specific. Particularly preferred among such expression vectorsare those inducible by environmental factors that are easy tomanipulate, such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophages, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papovaviruses, λ phage,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

In one embodiment, an isolated nucleic acid molecule of the invention orfragment thereof may be operably linked to an appropriate regulatorysequence, preferably a promoter such as the phage lambda PL promoter,promoters from T3, T7 and SP6 phages, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs and derivatives thereof, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the mature transcripts expressed by the constructswill preferably include a translation initiation codon (AUG) at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

As indicated above, the expression vectors will preferably include atleast one selectable marker. Such markers include dihydrofolatereductase (dhfr) or neomycin (neo) resistance for eukaryotic cellculture and tetracycline (tet) or ampicillin (amp) resistance genes forculturing in E. coli and other bacteria. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas Escherichia spp. cells (particularly E. coli), Bacillus spp. cells(particularly B. cereus, B. subtilis and B. megaterium), Streptomycesspp. cells, Salmonella spp. cells (particularly S. typhimurium) andXanthomonas spp. cells; fungal cells, including yeast cells such asSaccharomyces spp. cells; insect cells such as Drosophila S2, SpodopteraSf9 or Sf21 cells and Trichoplusa High-Five cells; other animal cells(particularly mammalian cells and most particularly human cells) such asCHO, COS, VERO, HeLa, Bowes melanoma cells and HepG2 and other livercell lines; and higher plant cells. Appropriate culture media andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A and pNH46A, available fromStratagene; pcDNA3 available from Invitrogen; and pGEX, pTrxfus,pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540 and pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1, pBK and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Other suitable vectors will bereadily apparent to the skilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3, T7 andSP6 phage promoters, the gpt promoter, the lambda PR and PL promotersand the trp promoter. Suitable eukaryotic promoters include the CMVimmediate early promoter, the HSV thymidine kinase promoter, the earlyand late SV40 promoters, the promoters of retroviral LTRs, such as thoseof the Rous sarcoma virus (RSV), and metallothionein promoters, such asthe mouse metallothionein-I promoter.

Introduction of the genetic constructs into the host cells can beeffected by a variety of methods, such as calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,nucleic acid-coated microprojectile bombardment or other methods. Suchmethods are described in many standard laboratory manuals, such as Daviset al., Basic Methods In Molecular Biology (1986).

Thus, the invention further provides, in an additional embodiment, amethod of ligating an amplified nucleic acid molecule into a vector withincreased efficiency. Such methods may comprise one or more steps, suchas (a) forming a mixture comprising one or more of the above-describednucleic acid molecules and one or more polymerase inhibitors; and (b)ligating the nucleic acid molecules into one or more of theabove-described vectors to form one or more genetic constructs.Analogously, the invention also provides methods suitable for cloning anucleic acid molecule, such as those described above, into one or moreof the above-described vectors. An exemplary method may comprise (a)forming a mixture comprising the nucleic acid molecules to be cloned,the cloning vectors and one or more polymerase inhibitors; and (b)ligating the nucleic acid molecules into one or more of theabove-described vectors to form one or more genetic constructs. In anadditional embodiment, the invention provides a further method ofcloning nucleic acid molecules, such as those described above, into oneor more vectors comprising: (a) forming a mixture comprising the nucleicacid molecules to be cloned, one or more polymerase inhibitors and oneor more of the above-described restriction endonucleases; and (b)ligating the nucleic acid molecules into one or more of theabove-described vectors to form one or more genetic constructs. In anadditional embodiment, the invention provides a further method ofcloning nucleic acid molecules, such as those described above, into oneor more vectors comprising: (a) forming a mixture comprising the nucleicacid molecules to be cloned, one or more polymerase inhibitors and oneor more of the above-described recombination proteins; and (b) ligatingthe nucleic acid molecules into one or more of the above-describedvectors to form one or more genetic constructs.

According to the invention, the mixture formed in the steps (a) of theabove-described methods may further comprise one or more additionalcomponents, including but not limited to one or more of theabove-described polypeptides having polymerase activity, one or moredNTPs or ddNTPs, one or more polypeptides having reverse transcriptaseactivity, one or more buffer salts, and the like. In one of the aboveaspects of the invention, the polypeptides having polymerase activityand the one or more restriction endonucleases or one or morerecombination proteins may be added to the mixture simultaneously. Inanother of the above aspects, the polymerases and endonucleases orrecombinases may be added sequentially, in any order. These methods ofthe invention may also further comprise one or more additional steps,such as the transformation of one or more of the genetic constructsformed by these methods into one or more of the above-described hostcells.

These methods of the invention may be advantageously used to clone orligate any nucleic acid molecule, which may be an amplified nucleic acidmolecule, into a vector and/or host cell. Thus, the invention alsoprovides nucleic acid molecules cloned by such methods, and host cellsproduced by being transformed with the above-described cloned nucleicacid molecules according to the methods of the invention.

Polymerase Inhibitors

As described above, the methods of the invention (particularly thecloning and ligation methods) advantageously utilize one or moreinhibitors of the polymerase activity of the polypeptides used toamplify the nucleic acid molecules. As used herein, an “inhibitor” of apolymerase is defined as any compound, composition or combinationthereof that inactivates or reduces the activity of a polypeptide havingnucleic acid polymerase activity, reversibly or irreversibly. Inparticular, inhibitors of a polymerase as used in the present inventionwill, upon contact with or binding to the polymerase polypeptide, reducethe activity of the polypeptide to no greater than about 70%, 60%, 50%,40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% 1% or 0.1%, of theactivity of a polypeptide having polymerase activity (such as thosedescribed above) that has not been contacted with the inhibitor. As apractical matter, whether a particular inhibitor reduces the activity tono greater than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1% or 0.1%, of the activity of an uninhibitedpolypeptide having polymerase activity, may be determined by measuringthe unit activity (by the methods described above and others that willbe familiar to one of ordinary skill) of the polymerase in the presenceand absence of various concentrations of the inhibitor.

A variety of inhibitors are suitable for use in the present methods.Included among these inhibitors are antibodies that bind to theabove-described polypeptides having polymerase activity (such asanti-Taq antibodies, anti-Tne antibodies, anti-Tma antibodies oranti-Pfu antibodies), and fragments thereof (such as Fab or Fab′₂fragments). Such antibodies may be polyclonal or monoclonal, and may beprepared in a variety of species according to methods that arewell-known in the art. See, for instance, Sutcliffe, J. G., et al.,Science 219:660-666 (1983); Wilson et al., Cell 37: 767 (1984); andBittle, F. J., et al., J. Gen. Virol. 66:2347-2354 (1985). Antibodiesspecific for any of the above-described polymerases, such as anti-Taqantibodies, anti-Tne antibodies, anti-Tma antibodies and anti-Pfuantibodies, can be raised against the intact polymerase polypeptide orone or more antigenic polypeptide fragments thereof. These polypeptidesor fragments may be presented together with a carrier protein (e.g.,albumin) to an animal system (such as rabbit or mouse) or, if they arelong enough (at least about 25 amino acids), without a carrier.

As used herein, the term “antibody” (Ab) may be used interchangeablywith the terms “polyclonal antibody” or “monoclonal antibody” (mAb),except in specific contexts as described below. These terms, as usedherein, are meant to include intact molecules as well as antibodyfragments (such as, for example, Fab and F(ab′)₂ fragments) which arecapable of specifically binding to a polypeptide having polymeraseactivity (such as a thermostable DNA polymerase or a reversetranscriptase) or a portion thereof.

The anti-polymerase antibodies used in the methods of the presentinvention may be polyclonal or monoclonal, and may be prepared by any ofa variety of methods (see, e.g., U.S. Pat. No. 5,587,287). For example,polyclonal antibodies may be made by immunizing an animal with one ormore polypeptides having polymerase activity or portions thereof (e.g.,one or more thermostable DNA polymerases such as Tag, Tne, Tma or Pfupolymerase) according to standard techniques (see, e.g., Harlow, E., andLane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press (1988); Kaufman, P. B., et al., In:Handbook of Molecular and Cellular Methods in Biology and Medicine, BocaRaton, Fla.: CRC Press, pp. 468-469 (1995)). Alternatively,anti-polymerase monoclonal antibodies (or fragments thereof), such asanti-DNA polymerase antibodies (e.g., anti-Taq, anti-Tne, anti-Tma oranti-Pfu antibodies) to be used in the present methods may be preparedusing hybridoma technology that is well-known in the art (Köhler et al.,Nature 256:495 (1975); Köhler et al., Eur. J. Immunol. 6:511 (1976);Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In:Monoclonal Antibodies and T-Cell Hybridomas, New York: Elsevier, pp.563-681 (1981); Kaufman, P. B., et al., In: Handbook of Molecular andCellular Methods in Biology and Medicine, Boca Raton, Fla.: CRC Press,pp. 444-467 (1995)).

In yet another approach, antibodies capable of binding to one or morepolypeptides having polymerase activity, or fragments thereof, may beused to remove the polypeptides having polymerase activity, thuspreventing the polymerase from having an adverse effect on cloning ofthe amplified molecule. In such a procedure, antibodies (or fragmentsthereof) specific for the polymerase may be used to remove thepolymerase from the reaction. Alternatively, the anti-polymeraseantibody may be used to inhibit/inactivate the polymerase and a secondantibody specific for the anti-polymerase antibody can be used to removethe inactivated polymerase.

It will be appreciated that Fab, F(ab′)₂ and other fragments of theabove-described antibodies may be used in the methods described herein.Such fragments are typically produced by proteolytic cleavage, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)₂ fragments). Polymerase-binding antibody fragments may also beproduced through the application of recombinant DNA technology orthrough synthetic chemistry.

Alternatively, antibodies directed against one or more of theabove-described polypeptides having polymerase activity, which may beused to inhibit the activity of residual polymerases in the reactionmixture following amplification of nucleic acid molecules, may beobtained commercially for example from Life Technologies, Inc.(Rockville, Md.), Boehringer Mannheim (Indianapolis, Ind.) and Sigma(St. Louis, Mo.).

In addition to antibodies, other compounds that are suitable asinhibitors for use in the present methods include chemicals, which maybe synthetic or naturally occurring (such as α-amanatin, polyethyleneglycol, dimethylsulfoxide, formamide, dimethylformamide, urea,pyrophosphate, acetic anhydride and diethylpyrocarbonate), antibiotics(such as actinomycin-D), heavy metals (such as compounds containingnickel (particularly Ni⁺⁺-containing salts) or copper (particularlyCu⁺⁺-containing salts)), acids (such as digallic acid, aurochloric acid,phosphonoformate and podoscyphic acid), metal chelators (such as EDTA),nucleotide analogues (such as peptide nucleic acid (PNA) and2-(p-n-butylanilino)-dATP), sulfhydryl reagents (such asn-ethylmaleimide or iodoacetic acid), anionic detergents (such as sodiumdodecylsulfate), polyanions (such as spermidine), captan((N-[trichloromethyl]-thio)-4-cyclohexene-1,2-dicarboximide), acidicpolysaccharides (such as dextran sulfate and heparin), a binding proteinor peptide, and combinations thereof. However, it will be understood bythe skilled artisan that any compound, natural or synthetic, thatinhibits or inactivates the polymerase activity of a polypeptideaccording to the above parameters may be advantageously used in themethods of the present invention.

In use, the one or more inhibitors function to increase the efficiencyof cloning of the above-described amplified, synthesized or digestednucleic acid molecules. It is thought that such an advantage is due tothe action of the inhibitors to prevent or inhibit modification of oneor more of the termini (3′ and/or 5′) of the amplified, synthesized ordigested nucleic acid molecules; it will be understood, however, thatregardless of the mechanism of action the one or more inhibitorsfunctions to provide increased efficiency of cloning of the amplified,synthesized and digested nucleic acid molecules.

Compositions

In an another embodiment, the present invention is directed tocompositions which may be used, for example, in the methods of thepresent invention to clone an amplified nucleic acid molecule.Compositions of the invention may comprise one or more components, whichmay be present in solution or in solid form, and which may be formulatedat working concentrations or in solutions of higher concentration (forexample, 2×, 2.5×, 5×, 10×, 20×, 25×, 50×, 100×, 250×, 500×, 1000× andthe like).

A preferred composition of the invention comprises one or more of theabove-described restriction endonucleases and one or more of theabove-described polymerase inhibitors. These restriction endonucleasesand polymerase inhibitors may be present at the working concentrationsnoted above, or at higher than working concentrations, and may each bepresent at different concentrations. In particularly preferredcompositions of the invention, the restriction endonucleases andpolymerase inhibitors are stable upon storage, such that thecompositions themselves may be stored for extended periods of timewithout losing activity. As noted above, the term “stable” as usedherein means that the restriction endonucleases and polymeraseinhibitors that make up the present compositions retain at least 70%,preferably at least 80%, and most preferably at least 90%, of theiroriginal enzymatic activity (in units) after the composition containingthe enzyme has been stored for at least four weeks at a temperature ofabout 20-25° C., at least one year at a temperature of about 4° C. or atleast 2 years at a temperature of −20° C.

Alternative compositions of the invention may comprise one or morepolymerase inhibitors, such as those described above, and may optionallyfurther comprise one or more additional components, for example, one ormore DNA-modifying enzymes or combinations thereof (such as ligases,topoisomerases, kinases, phosphatases, nucleases, endonucleases,terminal deoxynucleotidyl transferases, etc.).

The compositions of the invention may additionally comprise one or morenucleic acid molecules (including amplified nucleic acid molecules orfragments or derivatives thereof), one or more nucleotides (includingdNTPs, ddNTPs and/or rNTPs), one or more detergents (including TRITONX-100®, Nonidet P-40 (NP-40), Tween 20, Brij 35, sodium deoxycholate orsodium dodecylsulfate), one or more enzyme cofactors and/or one or moresuitable buffers (such as TRIS, phosphate salts (such as sodiumphosphate (mono- or dibasic) and potassium phosphate), sodiumbicarbonate, sodium acetate, HEPES, and the like). Combinations ofammonium sulfate, one or more magnesium salts (such as magnesiumchloride or magnesium sulfate), one or more manganese salts (such asmanganese sulfate) and potassium chloride (or other salts), may also beused in formulating the compositions ofthe present invention. A smallamount of a salt of ethylenediaminetetraacetate (EDTA) may also be added(preferably about 0.1 millimolar), although inclusion of EDTA does notappear to be essential to the function or stability of the compositionsof the present invention. Other components that may advantageously beadded to the present compositions to facilitate their use in cloning ofamplified nucleic acid molecules will be apparent to the skilledartisan.

Following formulation, the present compositions may be filtered througha low protein-binding filter unit that is available commercially (forexample from Millipore Corporation, Bedford, Mass.) and stored untiluse. To reduce component denaturation, storage of the presentcompositions is preferably in conditions of diminished light, e.g., inamber or otherwise opaque containers or in storage areas with controlledlow lighting. The compositions of the present invention are unexpectedlystable at ambient temperature (about 20°-25° C.) for about 4-10 weeks,are stable for at least one year upon storage at 4° C., and for at leasttwo years upon storage at −20° C. Surprisingly, storage of thecompositions at temperatures below freezing (e.g., −20° C. to −70° C.),as is traditional with stock solutions ofbioactive components, is notnecessary to maintain the stability of the compositions of the presentinvention.

Kits

In another embodiment, the invention relates to kits for cloning anamplified nucleic acid molecule. Kits according to the present inventionmay comprise a carrier means, such as a box, carton, tube or the like,having in close confinement therein one or more containers, such asvials, tubes, ampules, bottles and the like. A first container in thepresent kits may contain, for example, one or more of theabove-described polymerase inhibitors. The kits of the invention mayfurther comprise one or more additional containers containing one ormore additional reagents and compounds, such as one or more polypeptideshaving polymerase activity, one or more primers, one or more nucleotides(such as dNTPs, ddNTPs and/or rNTPs), one or more polypeptides havingreverse transcriptase activity, one or more nucleic acid-modifyingenzymes (such as topoisomerases, ligases, phosphatases, etc.), one ormore vectors, one or more host cells (particularly one or more of theabove-described host cells and most particularly one or moretransformation-competent host cells), one or more restrictionendonucleases, and one or more recombination proteins. Additional kitsof the invention may comprise one or more of the above-describedcompositions of the invention. These kits and their components arepreferably stable upon storage according to the above-describedparameters of stability, and may be advantageously used to clone anucleic acid molecule, preferably an amplified nucleic acid molecule,according to the methods of the invention.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Example 1 Inhibition of Polymerase Activity Facilitates Cloningof Amplified Nucleic Acid Molecules

In initial experiments, the amount of polymerase activity remaining in aPCR reaction mixture after amplification was determined. The activity ofTaq DNA polymerase remaining after 30 cycles of PCR was assayed directlyby the standard unit assay (Innis, M. A., et al., Proc. Natl. Acad. Sci.USA 85:9436-9440 (1988); Gelfand, D. H., in: Current Communications inMolecular Biology: Polymerase Chain Reaction, Ehrlich, H., et al., eds.,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, pp. 11-17(1989)), using several sources ofpolymerase: Native Taq (LifeTechnologies, Inc.), Elongase enzyme mix (Life Technologies, Inc.) andAmpliTaq (Perkin-Elmer). The results of these experiments indicated thatafter 30 cycles of PCR, 60-96% of the initial Taq activity remained(data not shown), which was sufficient Taq activity to perform another30 cycles of PCR. These findings therefore confirmed those previouslyreported (Bennett, B. L., and Molenaar, A. J., BioTechniques 16(1):36-37(1994).

To determine if elimination of this polymerase activity facilitatedcloning of the amplification products, a series of experiments wasconducted. PCR primers were designed to amplify out a 664-bp fragmentwithin the pPROEX-CAT cloning vector (Life Technologies, Inc.) whichencodes a gene for chloramphenicol acetyl transferase (CAT) and conferschloramphenicol resistance on transformants containing the gene. Theupstream (5′) primer was located near an EcoRI site in the CAT codingregion and the downstream (3′) primer bound to a region 3′ to the CATgene past a unique HindIII site. The experiments consisted of Taqpolymerase-mediated amplification of the plasmid insert, followed byvarious methods of Taq removal prior to digestion of the termini of thePCR products with EcoRI and HindIII. Because of extra nucleotidesequence contributions from the primer design, the actual PCR productwas 664 bp which generated a 522-bp fragment after digestion. Thedigested PCR product was then gel purified by electroelution prior toquantitation and ligation into an ampicillin-resistant pPROEX-CATcloning vector that had been similarly digested with EcoRI and HindIII.This experimental design therefore provided for direct detection of thenumber of transformants (i.e., the number of ampicillin-resistant(“amp^(r)”) colonies) and whether the insert was correctly ligated,in-frame, into the vector in these transformants. Specifically, if theinsert was correctly ligated, the ampicillin-resistant bacterialcolonies would also be resistant to chloramphenicol (”cam^(r)”) in thepresence of IPTG, which would induce expression of the CAT gene in theinsert. This assay also provided a means of identifying whether theresidual Taq polymerase had partially or completely filled in therestriction enzyme-generated cohesive ends on the amplicon: partialfill-in of termini by Taq prior to ligation would shift the readingframe of the ligated insert and result in improper transcription of theCAT gene and loss of chloramphenicol resistance (i.e., colonies would beobserved that were amp^(r) but not cam^(r)).

To serve as a positive control for this experiment, the plasmidpPROEX-CAT was digested with EcoRI and HindIII prior to agarose gelelectrophoresis and purification of each of the two generated DNAfragments. The purified vector and insert fragments were religated in a1:1 molar ratio; this preparation served as a positive control sinceneither fragment was exposed to Taq DNA polymerase. The negative controlconsisted of a “vector-only” ligation reaction.

The 664-bp fragment was amplified using the pPROEX-CAT vector as atemplate in a series of amplification reactions such that the totalreaction volume was equivalent to 5 ml. After confirmation of successfulamplification of the 664-bp fragment by agarose gel analysis andEtBr-staining, all of the reactions were pooled to minimize samplevariation that may have taken place during PCR which could potentiallybias the cloning results. This pool was then resplit into severalaliquots to undergo post-PCR manipulations, designed to reduce oreliminate residual Taq polymerase prior to cloning, as follows:

a.) Ethanol precipitation: A 600 μl aliquot of the reaction pool wasprecipitated with ethanol in the presence of sodium acetate, followed bydigestion of the precipitate with EcoRI and HindIII in React 2 buffer.Following RE-digestion, the sample was extracted once withphenol/chloroform/isoamyl alcohol (25:24:1) followed by an additional100% chloroform extraction. The sample was then subjected toelectrophoresis on a 1% agarose gel, followed by EtBr staining andexcision of the 564-bp fragment. The DNA was recovered from the gelslice by electroelution, ethanol precipitated again, resuspended in TEbuffer or sterile water and then a portion of it was visuallyquantitated on an agarose gel by EtBr-staining compared to similarlystained standards.

b.) Gel Purification: A 600 μl aliquot of the PCR pool was subjected toagarose-gel electrophoresis and EtBr-staining and was recovered from thegel by using a GlassMAX procedure according to the manufacturer'sinstructions (Life Technologies, Inc.). The recovered DNA fragment wasdigested with EcoRI and HindIII as described above, and subjected to asecond round of gel purification as described above using electroelutionand subsequent visual quantitation.

c.) Phenol/chloroform extraction: A 600 μl aliquot of the PCR pool wasextracted twice using an equal volume of phenol/chloroform (49:1)followed by ethanol precipitation in the presence of sodium acetate. Theprecipitate was then digested, purified by electrophoresis, ethanolprecipitated and visually quantitated as described above.

d.) Anti-Taq Antibody treatment: A 600 μl aliquot of the PCR pool wasadded to 6 μl of TaqStart antibody (CloneTech). Per the manufacturer'srecommendations, this was an appropriate amount of antibody toinactivate 25-30 units of Taq, the amount that was initially present inthe amplification reaction mixture. The reaction was incubated at about20-25° C. for 40 minutes to allow the antibody to bind to the Taqenzyme. Following this incubation, the reaction mix was digested withEcoRI and HindIII in React 2 buffer and then ethanol precipitated asabove. The sample was then subjected to agarose gel electrophoresis,electroelution and visual quantitation as described above.

Following these various treatments and visual quantitation of theresulting inserts, the inserts were ligated into the pPROEX-CAT vector.Ligation reactions were set up at a 1:1 (10 ng insert:100 ng vector)molar ratio, and incubated for 12-18 hours at 12° C. using T4 DNA ligasein a 20 μl volume. The reactions were then diluted to 100 μl of sterilewater, and 5 μl of this dilution were added to 100 μl oftransformation-competent DH10B E. coli cells (Life Technologies, Inc.).Following transformation, various dilutions of the reaction (1 ml each)were plated out on LBamp and LBcam/IPTG plates (Life Technologies, Inc.)to determine the numbers of recombinant (amp^(r)) and CAT-expressing(cam^(r)) colonies. Results are shown in Table 1.

TABLE 1 Effect of Taq Removal on Cloning Efficiency. CorrectRecombinants Post-PCR Cloning Efficiency % Cam^(r) treatment Number ofAmp^(r) colonies (No. of Colonies) vector-only 13 7 (1)  (neg. control)vector + insert 4250 84 (3550) religation (pos. control) Taq Antibody1500 85 (1275) treatment Ethanol 700 22 (154)  precipitation GelPurification 495 100 (495)  Phenol/chloroform 2350 96 (2256) extraction

The results shown in Table 1 demonstrate that treatment of the PCRsample with anti-Taq antibody prior to restriction enzyme digestionresulted in a high number of transformants (amp^(r) colonies), with alarge proportion (85%) of these being cam^(r) indicating that thecorrect reading frame was maintained during ligation. These resultscompare favorably with the positive control and also with thephenol/chloroform extraction method, which has been shown previously toresult in higher post-PCR cloning efficiencies (Bennett, B. L., andMolenaar, A. J., BioTechniques 16(1):36-37 (1994), and which hasheretofore been the method of choice for reducing residual Taq activity.Ethanol precipitation of the PCR product followed by digestion andpurification resulted in a reduced number of cam^(r) colonies,consistent with previous reports that ethanol precipitation isinsufficient to remove residual Taq DNA polymerase activity (Bennett, B.L., and Molenaar, A. J., BioTechniques 16(1):36-37 (1994). Gelpurification ofthe PCR product to remove Taq DNA polymerase prior toRE-digestion resulted in 100% of the amp^(r) recombinants being cam^(r),but the overall number of transformants were the lowest compared to theother treatment groups. Furthermore, this double gel purification wasthe most time-consuming and inefficient method since it resulted inlarge losses of amplification product due to the manipulations involvedin two successive rounds of agarose gel purification.

Together, these data indicate that it is advantageous to use a Taqantibody to facilitate post-PCR cloning of amplified nucleic acidmolecules. Such an approach increases the efficiency and yield of clonesobtained from amplified nucleic acid molecules, both by decreasing thenumber of experimental manipulations that are used and by obviating theuse of potentially harmful organic solvents (phenol/chloroform) forextraction of residual Taq activity.

Example 2 Radioactive Assay of Efficiency of Cloning of AmplifiedNucleic Acid Molecules

To confirm the above results in a more sensitive assay, a radioactivemethod was employed. A 664-bp amplicon, which encodes the 3′ end of thechloramphenicol acetyl transferase (CAT) gene, was amplified using TaqDNA polymerase (5 units/100 μl) and the primers detailed in Example 1.For set-up of the radioactive assay, the amplification product (5 μlcontaining 0.25 units of Taq DNA polymerase at the start of PCR) wasincubated in the presence and absence of two different preparations ofanti-Taq antibodies (“antibody #1” and “antibody #2”; Life Technologies,Inc. (Rockville, Md.)), for 15 minutes at room temperature (20-25° C.)prior to digestion with EcoRI and HindIII at 37° C. for one hour. Alongwith the restriction enzymes, 20 uCi of ³²P-dATP was added to eachreaction to monitor potential fill-in of 3′-recessed termini by residualTaq DNA polymerase. As an additional positive control, three units ofKlenow fragment were added to one reaction to examine maximalincorporation of nucleotides. Replicate samples were spotted induplicate onto glass fiber filters followed by precipitation ofincorporated nucleotides in the presence of ice cold 10% TCA/0.1%pyrophosphate.

The results of these experiments are shown Table 2.

TABLE 2 Effects of Anti-Taq Antibodies on 3′ Terminal Fill-inIncorporation Treatment Before RE Digestion (cpm; replicates) TCAprecipitation blank (negative control) 588, 976 none 7521,7811,7182,6868RE digestion followed by Klenow treatment 51271,53619 (positive control)Antibody #1, 1 unit 896, 664 Antibody #1, 0.67 units 598, 660 Antibody#1, 0.33 units 732, 696 Antibody #2, 1 unit 600, 602 Antibody #2, 0.67units 472, 536 Antibody #2, 0.33 units 670, 956

These results indicate that Taq DNA polymerase does, indeed, catalyzethe incorporation of nucleotides into TCA-precipitable material. Bothantibody formulations inhibited incorporation of TCA-precipitable countsto approximately background (blank) levels.

To determine if these cpm were incorporated into the clonable fragment,the samples were subjected to agarose-gel electrophoresis followed byautoradiography. As shown in FIG. 1, radiolabel was incorporated intothe 522-bp band in samples that were not preincubated with the antibodyprior to restriction enzyme digestion. The intensity of this 522-bp bandon the x-ray film was greatly diminished in all samples that wereincubated with Taq antibody prior to digestion, in support of the datashown above in Table 2.

Together, these results indicate that inclusion of anti-Taq antibodiesin the reaction mixture prevents the residual polymerase activity fromfilling in the 3′ recessed termini in restriction enzyme-digestedamplified nucleic acid molecules.

Example 3 Cloning of Amplified Nucleic Acid Molecules

A 664-bp amplicon was amplified using Taq DNA polymerase (5 units/100μl) as described in Example 1. 50 μl of the amplification reaction (2.5units of Taq DNA polymerase) were removed and incubated with 3.3 units(10 μl) of anti-Taq antibody (Life Technologies, Inc.; Rockville, Md.)for seven minutes at room temperature (about 20-25° C.). Two additional50 μl aliquots were incubated with 10 μl of antibody dilution buffer toserve as the control reactions. Following this preincubation, all threereactions were incubated with 60 units each of EcoRI and HindIII in atotal volume of 120 μl in React-2 buffer for one hour at 37° C. Fiveunits of Klenow enzyme were added to one of the control reactions andincubated for an additional 10 minutes at 37° C. to examine how fillingin the 3′ recessed ends of the digested amplicon affects the number ofcolonies obtained in a cloning experiment. All three reactions mixtureswere ethanol precipitated and subjected to agarose gel electrophoresisand the 522-bp clonable DNA was gel purified using GlassMax as describedin Example 1. Following purification, the concentration of the threedifferent insert preparations was determined using a Kodak DigitalImaging Camera so that equivalent amounts of the purified 522-bp insertwere added to each ligation reaction.

The three inserts were each ligated into gel-purified pPROEXCAT that hadbeen digested with EcoRI and HindIII. One tenth of the ligation wastransformed into DH5α subcloning-competent E. coli cells (LifeTechnologies, Inc.), and the transformations were plated in triplicateonto LB plates containing 100 μg/ml ampicillin and X-gal, and onto LBplates containing 100 μg/ml ampicillin, 7.5 μg/ml chloramphenicol, X-galand IPTG. The chloramphenicol/IPTG platings were performed to assess thepercentage of cam^(r) recombinants, as an indication of the number ofin-frame ligations as described above. Results are shown in Table 3.

TABLE 3 Efficiency of Cloning of Amplified Nucleic Acid Molecules.Sample No. of amp^(r) colonies No. of cam^(r) colonies vector-only 17 16(neg. control) Klenow fill-in 155 31 (pos. control) no antibody 28 48anti-Taq antibody 1317 932 insert only 0 nd* (neg. control) *nd = notdetermined

These results demonstrate that the addition of Taq antibody to thereaction, prior to restriction enzyme-mediated generation of 3′ recessedtermini, augments the efficiency of cloning of the amplified inserts.Based on these findings, it is therefore desirable to add a polymeraseinhibitor, such as an anti-Taq antibody, to the reaction mixture priorto digestion of PCR products with restriction enzymes, in order toincrease the efficiency of cloning of the amplified nucleic acidmolecules.

Example 4 Simultaneous Treatment of Amplijied Nucleic Acid Moleculeswith Anti-Taq Antibodies and Restriction Enzymes

The results shown in Example 3 indicate that sequential addition ofpolymerase inhibitors and restriction endonucleases greatly increasesthe efficiency of cloning of amplified nucleic acid molecules. Todetermine if this sequential addition of these reagents was necessary,the cloning experiments described in Example 3 were repeated, exceptthat the anti-Taq antibody was added to the reaction simultaneously withthe EcoRI and HindIII restriction enzymes (i.e., no preincubation, as inExample 3, was performed). In addition, a 1:1 ratio of Taq and anti-Tagantibody (per the unit definition of the antibody noted above) wasemployed; all other experimental techniques were the same as in Example3.

Results of these experiments are shown in Table 4.

TABLE 4 Simultaneous Addition of Antibody and Restriction Enzymes.Sample Treatment No. of amp^(r) colonies No. of cam^(r) coloniesvector-only 4 5 (neg. control) Klenow fill-in 9 7 (pos. control) noantibody 10 8 anti-Taq antibody 741 520 pUC 19 transformation 30 0control* *Transformation/plating control to demonstrate no growth on thechloramphenicol plates with a vector which cannot support CATexpression. Not corrected back to transformation efficiency (cfu/μg).

These results confirm those of Example 3, indicating that treatment ofthe amplification reaction mixture with a polymerase inhibitor, such asan anti-Taq antibody, increases the efficiency of cloning of theamplified nucleic acid molecules. More importantly, these resultsindicate that the polymerase inhibitors and the restrictionendonucleases may be added to the reaction mixture simultaneously orsequentially, with equivalent results.

Example 5 Reduction of Taq DNA Polymerase-Mediated Artifacts byTreatment of PCR Reactions with Anti-Taq Antibodies

When T/A cloning is performed, amplified nucleic acid is mixed (withoutpurification) with the cloning vector and then ligation is performed andhost cells transformed. The efficiency of this cloning method can beextremely variable. One parameter affecting efficiency is the stabilityof the vector which has 3′ dT overhangs that are needed to get specificannealing with the amplified product. In order to investigate the effectof Taq and other components of the PCR reaction on the vector itselfduring ligation, the following experiment was done. A mock PCR reactionwas prepared that contained all the components of a normal reactionexcept template and primers. Thirty cycles of amplification wereperformed, and an aliquot of this PCR reaction which was equivalent tothat normally used was added to the vector either with or withoutanti-Taq antibodies (obtained from Life Technologies, Inc.; Rockville,Md.). The ligation reaction and transformation were then performedaccording to the manufacturer's instructions (Invitrogen). The screenfor transformants in this system was a determination of the lack ofLacZα complementation (presence ofwhite colonies) as described inSambrook, J., et al., Molecular Cloning, A Laboratory Manual, 2nd ed.,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989). In anideal situation, there should be no colonies of any kind with the vectoralone in the ligation; typically, however, there a very small number ofbackground colonies are observed (i.e., 1-5 white colonies and 2-20 bluecolonies). White colonies result when the expression of the LacZα isstopped by insertion of an amplified product, or in this case by someother mechanism (for example, exonuclease contamination that chews backthe ends of the vector). Blue colonies are presumed to arise fromreligation of the vector that has lost the 3′ dT overhang and isreligated resulting in the expression of the α peptide.

As shown in Table 5, the presence of untreated Taq DNA polymerase (inthe PCR reaction mixtures) in the samples containing ligated vectoralone resulted in dramatically increased background (white/greencolonies). This background was reduced substantially in the samples thatwere treated with anti-Taq antibody prior to ligation.

TABLE 5 Reduction of Taq-mediated Cloning Artifacts Using Anti-TaqAntibodies Sample No. of White Colonies No. of Blue Colonies Vector +Taq (PCR  41, 122 30, 13  reaction) Vector + Taq (PCR 1, 4 2, 42reaction) + antibody Vector alone 1, 0 2, 24

These results indicate that Taq may have a deleterious effect on thevector (e.g., modifying the 3′ and/or 5′ termini) and may account forthe large variability in efficiency seen with T/A cloning. Reduction ofthis artifact, for example by use of anti-Taq antibody according to themethods of the present invention, leads to decreased background (lowernumber of colonies) and reduced variation in cloning efficiency.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1-52. (canceled)
 53. A method for cloning one or more nucleic acidmolecules into one or more vectors comprising: (a) forming a mixturecomprising said nucleic acid molecules to be cloned, one or morepolymerase inhibitors and one or more restriction endonucleases; and (b)ligating said nucleic acid molecules into one or more vectors to formone or more genetic constructs.
 54. the method of claim 53, wherein themixture further comprises one or more polypeptides having polymeraseactivity.
 55. The method of claim 53, wherein the nucleic acid moleculesto be cloned are amplified or synthesized nucleic acid molecules. 56.The method of claim 53, further comprising transforming the one or moregenetic constructs into one or more host cells.
 57. The method of claim53, wherein the inhibitors and the restriction endonucleases are addedsimultaneously.
 58. The method of claim 53, wherein the inhibitors andthe restriction endonucleases are added sequentially.
 59. A method ofligating and amplified or synthesized nucleic acid molecule into avector with increased efficiency, comprising: (a) forming a mixturecomprising the amplified or synthesized nucleic acid molecules and oneor more polymerase inhibitors; and (b) ligating the nucleic acidmolecules into one or more vectors to form one or more geneticconstructs.
 60. The method of claim 59, wherein the mixture furthercomprises one or more polypeptides having polymerase activity.
 61. Themethod of claim 59, further comprising transforming the one or moregenetic constructs into one or more host cells.
 62. A compositioncomprising one or more restriction endonucleases and one or morepolymerase inhibitors.
 63. The composition of claim 62 wherein the oneor more inhibitors are selected from the group consisting of an antibodyor a fragment thereof, a chemical compound, an antibiotic, a heavymetal, an acid, a metal chelator, a nucleotide analogue, a sulfhydrylreagent, an anionic detergent, a polyanion,captan((N-[trichloromethyl]-thio)-4-cyclohexene-1,2-dicarboximide), anacidic polysaccharide, a binding protein or peptide, and combinationsthereof.
 64. The composition of claim 63, wherein the inhibitor is anantibody or fragment thereof.
 65. The composition of claim 64, whereinthe antibody is selected from the group consisting of an anti-Taqantibody, an anti-Tne antibody, an anti-Tma antibody, an anti-Pfuantibody and fragments thereof.
 66. The composition of claim 62, furthercomprising one or more polypeptides having polymerase activity.
 67. Thecomposition of claim 62, further comprising one or more nucleic acidmolecules.